Development and validation of genome-informed and multigene-based qPCR and LAMP assays for accurate detection of Dickeya solani: a critical quarantine pathogen threatening the potato industry

Abstract

Dickeya solani one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the world's most important food crops. The diagnostics methods are critical in monitoring the latent infection for international trade of potato seed tubers and in implementation of control strategies. Our study employed a whole-genome comparative approach, identifying unique target gene loci (LysR and TetR family of transcriptional regulators gene regions) and designing loop-mediated isothermal amplification (LAMP) and a multi-gene-based multiplex TaqMan qPCR assays for specific detection and differentiation of D. solani. Both methods underwent meticulous validation with extensive inclusivity and exclusivity panels, exhibiting 100% accuracy and no false positives or negatives. The LAMP method demonstrated the detection limit of 100 fg and 1 CFU per reaction using pure genomic DNA and crude bacterial cell lysate, respectively. The qPCR detection limit was 1 pg, 100 fg and 10 fg with quadplex, triplex, and singleplex, respectively. None of the assays were impacted by any inhibitory or competitive effects after adding host DNA (in qPCR) or crude lysate (in LAMP). The assays proved robust and reproducible in detecting the target pathogen in infected samples, with the LAMP assay being field-deployable due to its simplicity and rapid results acquisition within approximately 9 min. The reproducibility was confirmed by performing the assay in two independent laboratories. These assays offer a robust, rapid, and reliable solution for routine testing, with applications in phytosanitary inspection, disease diagnosis, and epidemiological studies.IMPORTANCEDickeya solani, one of the most aggressive soft rot causing bacteria and a quarantine pathogen, poses a severe threat to food security by causing substantial economic losses to the potato industry. Accurate and timely detection of this bacterium is vital for monitoring latent infections, particularly for international trade of potato seed tubers, and for implementing effective control strategies. In this research, we have developed LAMP and multi-gene-based multiplex TaqMan qPCR assays for specific detection of D. solani. These assays, characterized by their precision, rapidity, and robustness, are crucial for distinguishing D. solani from related species. Offering unparalleled sensitivity and specificity, these assays are indispensable for phytosanitary inspection and epidemiological monitoring, providing a powerful tool enabling management of this threatening pathogen.

Keywords: Dickeya solani; blackleg; detection; diagnostics; isothermal amplification; potato; quarantine plant pathogens.

Fig 1

Ring plot representing the position of the two unique coding sequences TetR and LysR family transcriptional regulators used for the specific detection of Dickeya solani. The layers show the multiple genome alignment of five D. solani strains, followed by the other six members within the Dickeya genus and other bacterial species causing diseases to potato. From innermost to the outer, the layers indicate the genome coordinates (mega base pairs; mbp), the GC content (zigzag black line), and the GC skew (zigzag purple+ / green-) of the reference genome D. solani type strain D s0432-1ᵀ. The color-coded rings illustrate from inwards the BLASTn pairwise comparison of D. solani D s0432-1ᵀ (NZ_CP017453.1), D. solani IPO2222 (NZ_CP015137.1), D. solani PPO9019 (NZ_CP017454.1), D. solani RNS 08.23.3.1.A (NZ_CP016928.1), and D. solani IFB 0099 (NZ_CP024711.1), position of the two targets coding sequences, LysR and TetR family transcriptional regulators conserved across all D. solani strains (highlighted and labeled with fuchsia and red colors, respectively), D. chrysanthemi Ech1591ᵀ (NC_012912), D. dadantii 3937 (NC_014500), D. parazeae Ech586 (NC_013592; formerly known as D. zeae), Musicola paradisiaca Ech703 (NC_012880; previously called as D. paradisiaca), D. dianthicola RNS04.9ᵀ (NZ_CP017638.1), D. fangzhongdai DSM101947ᵀ (NZ_CP025003), D. aquatica 174/2ᵀ (NZ_LT615367), P. carotovorum PCC21 (NZ_018525), Erwinia amylovora CFBP 1430ᵀ (NC_013961), Ralstonia pseudosolanacearum GMI 1000ᵀ (NC_003295), and Clavibacter sepedonicus ATCC 33113ᵀ (NC_10407). The image was created using the BLAST Ring Image Generator (BRIG) v 0.95 (49).

Fig 2

Specificity validation of loop-mediated isothermal amplification (LAMP) for D. solani detection. Fluorescence detection under UV light. (A) Inclusivity assay with all D. solani strains: Tube one contains the positive control (genomic DNA of D. solani A5581), Tubes 2–11 contain genomic DNA of D. solani strains A5582, A6288, A6289, A6291, A6292, and A6294–A6298, and Tube N is the non-template control (NTC; water). (B) Exclusivity assay with genomic DNA from 13 representative bacterial strains from the exclusivity panel: Tube 1 contains D. solani (A5581); Tubes 2–15 contain D. dianthicola, D. paradisiaca, P. carotovorum, D. zeae, P. brasiliense, D. chrysanthemi, P. carotovorum, Pantoea sp., D. dadantii, E. amylovora, Klebsiella sp., P. betavasculorum, P. odoriferum, and healthy S. tuberosum (negative control); N is water (NTC).

Fig 3

(A–H): Sensitivity assays of loop-mediated isothermal amplification (LAMP) for specific detection of D. solani. (A–D) LAMP sensitivity assay; Lane M, 100 bp ladder, Lanes 1–8, 10-fold serially diluted purified genomic DNA of D. solani from 10 ng to 1 fg.; Lane N, negative template control (NTC, water). (E–H) Spiked LAMP sensitivity assay; lane M, 100 bp ladder; Lanes 1–8 10-fold serially diluted (1 ng to 1 fg) purified genomic DNA of D. solani plus 1 µL of host DNA (10 ng/µL) added to each reaction; Lane N, negative template control (NTC, water). (A and E) Rotor-Gene Q amplification of sensitivity assay and spiked sensitivity assay; (B and F) visual detection of LAMP after adding SYBR Green I; (C and G) tubes under UV; (D and H) amplified LAMP products on 2% agarose gel. (I–L): Visual detection of sensitivity assays of D. solani heat-killed cells using LAMP assay. (I and J) Tubes 1–9 10-fold dilution series consisting of 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10, and 1 CFU/mL of heat-killed cells of D. solani and a non-template control (tube N). (K and L) Tubes 1–9 10-fold dilution series consisting of 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10, and 1 CFU/mL of heat-killed cells of D. solani spiked with host lysate, and a no-template control (tube N). Products were detected after adding 3 µL SYBR Green I under visual light, where a positive result changes from orange to green (I and K), or ultraviolet light, where a positive result show fluorescence (J and L).

Fig 4

Standard curves and graphs for TaqMan-qPCR generated using ten-fold serial diluted Dickeya solani genomic DNA (10 ng to one fg). (A and C) Multiplex TaqMan-qPCR, (D and E) Single TaqMan-qPCR with 10-fold serially diluted genomic DNA; and (B) Multiplex TaqMan qPCR 10-fold serially diluted genomic DNA mixed with host plant DNA. The orange, green, yellow and crimson channels correspond to the different reported dyes Red –BHQ2, 6-FAM (495/520), HEX (535/554), and Quasar705-IBQ3 (excitation/emission spectra in nm), respectively. A1/A2/A3/A4-multiplex TaqMan qPCR generated by multiplexing Dso-wF1/wR1/Dso-P1, Dso-wF2/wR2/Dso-P2, DICg-wF1/wR1/DICg-P, and UIC-wF/wR/UIC-P primer/probe sets. B1/B2/B3/B4-spiked multiplex TaqMan qPCR generated by multiplexing Dso-wF1/wR1/Dso-P1, Dso-wF2/wR2/Dso-P2, DICg-wF1/wR1/DICgP, UIC-wF/wR/ UIC-P primer/probe sets; the spiked assay was done by adding 1 µL of healthy potato DNA extracted from tubers to each 10-fold serially diluted D. solani DNA. C1/C2/C3-multiplex TaqMan qPCR was generated by multiplexing Dso-wF1/wR1/Dso-P1, Dso-wF2/wR2/Dso-P2, and DICg-wF1/wR1/DICg-P primer/probe sets. (D) Single TaqMan qPCR with Dso-wF1/wR1/Dso-P1 primer set in the reaction mix. (E) Single TaqMan qPCR with Dso-wF2/wR2/Dso-P2 probe and primer set. X axis represents the number of cycles, and Y axis-normalized fluorescence. The C_T values are average of three replicates ±SD. Slopes (Y = threshold cycles (Ct) of target DNA detected), R² (correlation coefficient), and E (amplification efficiency).

Fig 5

Validation of Dickeya solani-specific loop-mediated isothermal amplification (LAMP) assay with plant samples naturally infected with other soft rot bacteria. Pectobacterium. brasiliense PL68 (PS32F), PL109 (PS33), and PL107 (PS60); P. parmentieri PL124 (PS38), and PL75 (PS63); D. dianthicola PL125 (PS1), PL127 (PS10), and PL122 (PS66); P. aroidearum PL66 (PS2); P. carotovorum PL73, and healthy potato plant sample. (A) Real-time amplification plot, (B) Melting curve. A positive control includes DNA from D. solani (A6295; positive amplification shown in both figures).